Mosquitoes Use Vision to Associate Odor Plumes with Thermal Targets Floris van Breugel, Jeff Riffell, Adrienne Fairhall, Michael H. Dickinson  Current Biology  Volume 25, Issue 16, Pages 2123-2129 (August 2015) DOI: 10.1016/j.cub.2015.06.046 Copyright © 2015 Elsevier Ltd Terms and Conditions

Current Biology 2015 25, 2123-2129DOI: (10.1016/j.cub.2015.06.046) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 1 Wind Tunnel, CO2 Plume, and Example Flight Trajectories (A) Wind tunnel used in our experiments. Color borders indicate top, side, and upwind views used in subsequent panels. (B) Heatmap of a turbulent-flow, particle-diffusion model of the CO2 plume based on 65 measurements in the wind tunnel; see the Experimental Procedures and Figure S1 for details. The white dot indicates a mosquito, drawn to scale. (C) Example flight trajectory in clean air. The two colored arrowheads show synchronized points across side and top views. The spacing between the points (33-Hz intervals) indicates the animal’s speed. (D) Example flight trajectory in the presence of a CO2 plume, showing the mosquitoes’ stereotypical behavior exploring the high-contrast object after sensing CO2. In both (C) and (D), an estimate of the animal’s instantaneous CO2 (or control) experience is plotted below and color-coded within the trajectories using the scale in (B). See also Figure S1 for additional trajectories and Movie S1 for animations. Current Biology 2015 25, 2123-2129DOI: (10.1016/j.cub.2015.06.046) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 2 CO2 Triggers Mosquitoes to Explore High-Contrast Dark Objects (A) Heatmap showing where female mosquitoes spent their time over a 3-hr period. The top panel shows a side view of the data. The bottom panel shows a top down view of the data over the altitude range indicated by the vertical pink line in the top panel. The right panel shows a photograph of the wind tunnel. (B) Same as (A), but in the presence of a CO2 plume. (C) Same as (A), but in the presence of a CO2 plume and with a black and a white visual object on the floor of the tunnel. (D) Same as (A), but with male mosquitoes in clean air. We did not find any qualitative differences in male mosquitoes’ behavior in the presence of a CO2 plume (not shown). (E) Relative flight activity, measured as the ratio of time mosquitoes spent flying in the presence of a CO2 or clean air plume compared to their prior activity. (F) The ratio of the total time mosquitoes spent near the object divided by the total time they spent elsewhere for CO2 and clean air conditions. Shading shows bootstrapped 95% confidence intervals of the mean. (G) Time elapsed between when mosquitoes left the plume (conservatively defined here as 401 ppm) and when they approached to within 3 cm of the object. (H) Example trajectories (top row, side view; bottom row, top-down view) that contributed to the histogram shown in (G), demonstrating the circuitous path many mosquitoes took from the plume to the object. Only the trajectory segments between plume exit (pink arrow) and object approach are shown. See also Figure S2 for a description of plume-tracking behavior and Figure S3. Current Biology 2015 25, 2123-2129DOI: (10.1016/j.cub.2015.06.046) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 3 Visual Stimuli Provide an Intermediate Cue, Linking Long-Range Olfactory Cues and Short-Range Heat Sensing (A) Photograph of the ITO-coated glass pad. (B) Measurements of the thermal plume created by the heated glass pads at altitudes of 0.5, 2.5, and 6.5 cm, colored orange, purple, and black, respectively. (C) Photographs and thermal images of the stimuli in the wind tunnel. (D) Mean fraction of trajectories that entered an 8 × 8 × 4 cm3 volume above and downwind of either the left or right object (see F). Shading indicates 95% confidence intervals. The letters at the top indicate significantly different groups (Mann-Whitney U test with Bonferroni correction, p = 0.01). (E) Mean preference index for the test object versus control object with 95% confidence intervals. Statistics were calculated as in (D). (F) Sample trajectory entering one of the test volumes (green) used in (D) and (E). The trajectory is color-coded red for the 2 s prior to when it entered the volume. (G) Spatial representation of preference index prior to when mosquitoes entered either test volume shown in (F). For each trajectory, we selected the segments 2 s prior to when they entered either volume, in addition to the portions spent inside the volumes (red region of the trajectory shown in F). We then calculated the preference index for each 2 × 2 cm2 rectangular region as the amount of time spent on the side of the wind tunnel of the test object compared to the control object, divided by their sum. We then calculated the mean preference index for each 2 × 2 cm2 region across all trajectories and its 95% confidence interval. Colors indicate preference index for regions where the 95% confidence interval was smaller than 0.5 (out of the total range of −1 to +1); the regions with higher uncertainty are shown in black. Blue or pink colors that are more saturated than the arrows on the scale bar represent regions where the mosquitoes showed a statistically significant preference for one side or the other. The average approach trajectory for all the mosquitoes in each trial is shown as a magenta line. Because the average approach trajectories to the two objects were indistinguishable, this line shows the average approach of all trajectories for simplicity. The light-green box shows a side view of the volumes shown in (F). The colored arrows indicate the altitudes at which the temperature of the air was measured in (B). The number of trajectories that approached the test (orange), control (blue), or both (black) objects is indicated in the top right of each panel. Current Biology 2015 25, 2123-2129DOI: (10.1016/j.cub.2015.06.046) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 4 Thermal Signature of a Human Arm Temperature downwind (0.4 m/s) of a human arm measured at two different ambient temperatures (orange and purple). Horizontal lines indicate the ambient temperatures. Current Biology 2015 25, 2123-2129DOI: (10.1016/j.cub.2015.06.046) Copyright © 2015 Elsevier Ltd Terms and Conditions